Polymer-conjugated MetAP2 inhibitors, and therapeutic methods of use thereof

ABSTRACT

One aspect of the invention provides polymer conjugated MetAP2 inhibitors. While not being bound by any particular theory, it is believed that coupling the MetAP2 inhibitory core via the linkers described herein provides compounds with superior efficacy to the parent small molecules and superior pharmacokinetic profiles. In one aspect of the invention, the polymer conjugated MetAP2 inhibitors are useful in methods of treating disease, comprising administering to a subject in need thereof a therapeutically effective amount of a polymer conjugated MetAP2 inhibitor.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/696,743, filed Nov. 7, 2012, now U.S. Pat. No.9,320,805, which is a 35 U.S.C. 371 NATL phase entry ofPCT/US2011/037857, filed May 25, 2011, which claims the benefit of, andpriority to, U.S. Provisional Patent Application Ser. No. 61/347,924,filed May 25, 2010.

BACKGROUND

Helmut Ringsdorf provided a theoretical framework for the design ofpolymer conjugates of small molecule drugs over thirty years ago (SeeRingsdorf, “Structure and Properties of Pharmacologically ActivePolymers”, J. POLYMER SCI.: Symposium No. 51, 135-153 (1975)). Whilemany conjugates have been synthesized and evaluated in animals, few haveprogressed to clinical trials and those trials have been largelydisappointing. The identification of polymer drug conjugates thatrepresent improvements over the parent small molecules remains an areaof active research.

Fumagillin is a small molecule which has been used as an antimicrobialand antiprotozoal agent. Its physiochemical properties and method ofproduction are well known (See U.S. Pat. No. 2,803,586 (Peterson, et al,incorporated herein by reference) and Turner, J. R. et al, TheStereochemistry of Fumagillin, Proc. Natl. Acad. Sci. 48, 733-735(1962)). The fermentation product, fumagillin, may be hydrolyzed toyield the alcohol fumagillol which in turn may be converted into variousderivatives including carbamoylfumagillol, MW 325. The synthesis andpreparation of carbamoylfumagillol and some small molecule derivativesare described in U.S. Pat. No. 5,166,172.

Fumagillin and related compounds are believed to exert their biologicaleffects through the inhibition of methionine aminopeptidase-2 (MetAP2),a metalloprotease. This enzyme removes N-terminal methionine fromnascent cellular proteins. (See Tucker, L. A., et at “Ectopic Expressionof Methionine Aminopeptidase-2 Causes Cell Transformation and StimulatesProliferation”, Oncogene 27, 3967 (2008).)

Carbamoylfumagillol and derivatives as well as other inhibitors ofMetAP2 have shown therapeutic benefits in preclinical and clinicalstudies. These compounds inhibit cell proliferation and angiogenesis asdescribed in U.S. Pat. No. 5,166,172 (Kishimoto, et al, incorporatedherein by reference). One of these derivatives,chloroacetylcarbamoylfumagillol (TNP-470) has been extensively studied.(See H. Mann-Steinberg, et al., “TNP-470: The Resurrection of the FirstSynthetic Angiogenesis Inhibitor”, Chapter 35 in Folkman and Figg,Angiogenesis: An Integrative Approach from Science to Medicine, SpringerNY (2008).) TNP-470 has shown activity against many cancers, includinglung cancer, cervical cancer, ovarian cancer, breast cancer, and coloncancer. Because of dose-limiting neurotoxicity, TNP-470 has been testedusing multiple dosing regimens, but these attempts to limit its toxicityhave been unsuccessful. Thus, TNP-470 has been found to be too toxic forhuman use. With few exceptions, unacceptable weight loss or failure togain weight was observed in animals receiving TNP-470. TNP-470 has ashort half-life and requires extended intravenous administration fortherapeutic use. A metabolite of TNP-470, carbamoylfumagillol has ahalf-life of 12 minutes in man. (See Herbst et al., “Safety andPharmacokinetic Effects of TNP-470, an Angiogenesis Inhibitor, Combinedwith Paclitaxel in Patients with Solid Tumors: Evidence for Activity inNon-Small-Cell Lung Cancer”, Journal of Clinical Oncology 20(22)4440-4447 (2002). In addition, fumagillin and its derivatives arehydrophobic and difficult to formulate.

Methionine aminopeptidase-2 (MetAP2), a metalloprotease, is an enzymethat processes N-terminal methionine from nascent cellular proteins.Inhibition of MetAP2 has been shown to block angiogenesis and suppresstumor growth in preclinical tumor models. Interestingly, fumagillin,chloroacetylcarbamoylfumagillol, carbamoylfumagillol and relatedcompounds have been shown to be inhibitors of MetAP2. (See Tucker, L.A., et al. “Ectopic Expression of Methionine Aminopeptidase-2 CausesCell Transformation and Stimulates Proliferation”, Oncogene 27, 3967(2008).)

SUMMARY

One aspect of the present invention relates to a compound orpharmaceutically acceptable salt thereof, comprising:

wherein, independently for each occurrence,

-   -   R₄ is H or C₁-C₆ alkyl;    -   R₅ is H or C₁-C₆ alkyl;    -   R₆ is C₂-C₆ hydroxyalkyl;    -   Z is —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L or        —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W;    -   AA₁ is glycine, alanine, or H₂N(CH₂)MCO₂H, wherein m is 2, 3, 4        or 5;    -   AA₂ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₃ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₄ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₅ is a bond, or glycine, valine, tyrosine, tryptophan,        phenylalanine, methionine, leucine, isoleucine, or asparagine;    -   AA₆ is a bond, or alanine, asparagine, citrulline, glutamine,        glycine, leucine, methionine, phenylalanine, serine, threonine,        tryptophan, tyrosine, valine, or H₂N(CH₂)_(m)CO₂H, wherein m is        2, 3, 4 or 5;    -   L is —OH, —O-succinimide, —O-sulfosuccinimide, alkoxy, aryloxy,        acyloxy, aroyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —NH₂,        —NH(C₂-C₆ hydroxyalkyl), halide or perfluoroalkyloxy;    -   Q is NR, O, or S;    -   X is M-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V;    -   M is a bond, or C(O);    -   J is a bond, or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl,        heteroaryl, NR, O, or S;    -   Y is NR, O, or S;    -   R is H or alkyl;    -   V is a bond or

-   -   R₉ is alkyl, aryl, aralkyl, or a bond; or R₉ taken together with        Y forms a heterocyclic ring;    -   R₁₀ is amido or a bond;    -   R₁₁ is H or alkyl;    -   W is a MetAP₂ inhibitor moiety or alkyl;    -   x is in the range of 1 to about 450;    -   y is in the range of 1 to about 30;    -   n is in the range of 1 to about 50;    -   p is 0 to 20;    -   q is 2 or 3;    -   r is 1, 2, 3, 4, 5, or 6; and    -   the compound has a molecular weight of less than about 60 kDa.

Another aspect of the present invention relates to a compound orpharmaceutically acceptable salt thereof, represented by Z-Q-X—Y—C(O)—W;wherein, independently for each occurrence,

-   -   Z is H₂N-AA₆-C(O)— or H;    -   AA₆ is alanine, asparagine, citrulline, glutamine, glycine,        leucine, methionine, phenylalanine, serine, threonine,        tryptophan, tyrosine, valine or H₂N(CH₂)_(m)CO₂H, wherein m is        2, 3, 4 or 5;    -   Q is NR, O, or S;    -   X is M-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V;    -   M is a bond, or C(O);    -   J is a bond, or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl,        heteroaryl, NR, O, or S;    -   Y is NR, O, or S;    -   R is H or alkyl;    -   V is a bond or

-   -   R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with        Y forms a heterocyclic ring;    -   R¹⁰ is amido or a bond;    -   R¹¹ is H or alkyl;    -   W is a MetAP2 inhibitor moiety;    -   p is 0 to 20;    -   q is 2 or 3; and    -   r is 1, 2, 3, 4, 5, or 6.

Another aspect of the invention relates to the use of a compound of theinvention to treat a disease (e.g., cancer) in a mammal in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows percentage weight change as a function of time for C57B1/6mice, injected initially with B16-F10 tumor cells (1×10⁵), to which oneof three polymer conjugates (dosed at 100 mg/kg, q4d) has beenadministered. Comparative data are included for TNP-470 (dosed at 30mg/kg, qod) and saline control.

FIG. 2 shows percentage weight change as a function of time for C57B1/6mice, injected initially with B16-F10 tumor cells (1×10⁵), to which apolymer conjugate (dosed at 100, 50 and 25 mg/kg, q4d) has beenadministered. Comparative data are included for TNP-470 (dosed at 30mg/kg, qod) and saline control.

FIG. 3 shows the change in tumor size as a function of time for nu/numice, injected initially with A549 tumor cells, to which one of threepolymer conjugates (dosed at 20 mg/kg, q4d) has been administered.Comparative data are included for TNP-470 (30 mg/kg, qod), a polymerwithout drug (100 mg/kg, q4d) and saline control.

FIG. 4 shows the change in body weight change as a function of time fornu/nu mice, injected initially with A549 tumor cells, to which one ofthree polymer conjugates (dosed at 20 mg/kg, q4d) has been administered.Comparative data are included for TNP-470 (30 mg/kg, qod), a polymerwithout drug (100 mg/kg, q4d) and saline control.

DETAILED DESCRIPTION

One aspect of the present invention relates to a compound orpharmaceutically acceptable salt thereof, comprising:

wherein, independently for each occurrence,

-   -   R₄ is H or C₁-C₆ alkyl;    -   R₅ is H or C₁-C₆ alkyl;    -   R₅ is C₂-C₆ hydroxyalkyl;    -   Z is —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L or        —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W;    -   AA₁ is glycine, alanine, or H₂N(CH₂)_(m)CO₂H, wherein m is 2, 3,        4 or 5;    -   AA₂ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₃ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₄ is a bond, or alanine, cysteine, aspartic acid, glutamic        acid, phenylalanine, glycine, histidine, isoleucine, lysine,        leucine, methionine, asparagine, proline, glutamine, arginine,        serine, threonine, valine, tryptophan, or tyrosine;    -   AA₅ is a bond, or glycine, valine, tyrosine, tryptophan,        phenylalanine, methionine, leucine, isoleucine, or asparagine;    -   AA₆ is a bond, or alanine, asparagine, citrulline, glutamine,        glycine, leucine, methionine, phenylalanine, serine, threonine,        tryptophan, tyrosine, valine, or H₂N(CH₂)_(m)CO₂H, wherein m is        2, 3, 4 or 5;    -   L is —OH, —O-succinimide, —O-.sulfosuccinimide, alkoxy, aryloxy,        acyloxy, aroyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —NH₂,        —NH(C₂-C₆ hydroxyalkyl), halide or perfluoroalkyloxy;    -   Q is NR, O, or S;    -   X is M-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V;    -   M is a bond, or C(O);    -   J is a bond, or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl,        heteroaryl, NR, O, or S;    -   Y is NR, O, or S;    -   R is H or alkyl;    -   V is a bond or

-   -   R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with        Y forms a heterocyclic ring;    -   R¹⁰ amido or a bond;    -   R¹¹ is H or alkyl;    -   W is a MetAP2 inhibitor moiety or alkyl;    -   x is in the range of 1 to about 450;    -   y is in the range of 1 to about 30;    -   n is in the range of 1 to about 50;    -   p is 0 to 20;    -   q is 2 or 3;    -   r is 1, 2, 3, 4, 5, or 6; and    -   the compound has a molecular weight of less than about 60 kDa.

In certain embodiments, P is C₁-C₆ alkyl. In certain embodiments, R₄ ismethyl. In certain embodiments, R₅ is C₁-C₆ alkyl. In certainembodiments, R₅ is methyl. In certain embodiments, R₆ is 2-hydroxyethyl,2-hydroxypropyl or 3-hydroxypropyl. In certain embodiments, R₆ is2-hydroxypropyl.

In other embodiments, the molecular weight is less than about 45 kDa. Inother embodiments, the molecular weight is less than about 35 kDa.

In certain embodiments, the ratio of x to y is in the range of about30:1 to about 3:1. In other embodiments, the ratio of x to y is in therange of about 20:1 to about 4:1. In certain embodiments, the ratio of xto y is in the range of about 15:1 to about 6:1. In certain embodiments,the ratio of x to y is about 15:1. In certain embodiments, the ratio ofx to y is about 11:1. In certain embodiments, the ratio of x to y isabout 6:1.

In certain embodiments, Z is —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L.

In certain embodiments, L is methoxy, ethoxy, pentafluorophenyloxy,phenyloxy, acetoxy, fluoride, chloride, methoxycarbonyloxy;ethoxycarbonyloxy, phenyloxycarbonyloxy, 4-nitrophenyloxy,trifluoromethoxy, pentafluoroethoxy, or trifluoroethoxy. In certainembodiments, L is 4-nitrophenyloxy.

In certain embodiments, Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W. In certain embodiments,AA₁ is glycine. In certain embodiments, AA₂ is glycine. In certainembodiments, AA₃ is glycine. In certain embodiments, AA₄ is glycine orphenylalanine. In certain embodiments, AA₅ is leucine, phenylalanine,valine or tyrosine. In certain embodiments, AA₆ is asparagine,citrulline, glutamine, glycine, leucine, methionine, threonine ortyrosine. In certain embodiments, AA₅-AA₆ is Leu-Cit, Leu-Gln, Leu-Gly,Leu-Leu, Leu-Met, Leu-Thr, Phe-Cit, Phe-Gln, Phe-Leu, Phe-Met, Phe-Thr,Val-Asn, Val-Cit, Val-Gln, Val-Leu, Val-Met, Val-Thr, Tyr-Cit, Tyr-Leu,or Tyr-Met. In certain embodiments, AA₁, AA₃ and AA₅ are glycine,valine, tyrosine, tryptophan, phenylalanine, methionine, leucine,isoleucine, or asparagine. In certain embodiments, AA₂, AA₄ and AA₆ areglycine, asparagine, citrulline, glutamine, glycine, leucine,methionine, phenylalanine, threonine or tyrosine. In certainembodiments, AA₂ is a bond; and AA₃ is a bond. In certain embodiments,AA₁ is glycine; AA₄ is phenylalanine; AA₅ is leucine; and AA₆ isglycine.

In certain embodiments, W is

wherein R₂ is —OH or methoxy; and R₃ is H, —OH or methoxy.

In certain embodiments, W is

In certain embodiments, W is

In certain embodiments, Q is NR. In other embodiments, Q is S.

In certain embodiments, J is NR. In other embodiments, J is((CH₂)_(q)Q)_(r). In other embodiments, J is C₅-C₈ cycloalkyl. Incertain embodiments, J is aryl.

In certain embodiments, Y is NR. In other embodiments, Y is S.

In certain embodiments, -Q-X—Y— is

V is:

or a bond;

-   -   R¹² is H or Me; or R taken together with R forms a piperidine        ring;    -   R¹¹ is H or Me; and    -   R¹³ taken together with R¹² forms a piperidine ring.

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, R₄ and R₅ are methyl; is 2-hydroxypropyl; Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ is abond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine; AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, R₄ and R₅ are methyl; is 2-hydroxypropyl; Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ is abond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine; AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, R₄ and R₅ are methyl; R₆ is 2-hydroxypropyl; Zis —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ isa bond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine, AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, -Q-X—Y— is a self-immolating linker thatreleases the MetAP2 inhibitor in the form of a carbamate derivative, asshown in the scheme below:

Another aspect of the present invention relates to a compound orpharmaceutically acceptable salt thereof, represented byZ-Q-X—Y—C(O)—W

-   -   wherein, independently for each occurrence,    -   Z is H₂N-AA₆-C(O)— or H;    -   AA₆ is alanine, asparagine, citrulline, glutamine, glycine,        leucine, methionine, phenylalanine, serine, threonine,        tryptophan, tyrosine, valine or H₂N(CH₂)mCO₂H, wherein m is 2,        3, 4 or 5;    -   Q is NR, O, or S;    -   X is M-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V;    -   M is a bond, or C(O);    -   J is a bond, or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl,        heteroaryl, NR, O, or S;    -   Y is NR, O, or S;    -   R is H or alkyl;    -   V is a bond or    -   R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with        Y forms a heterocyclic ring;    -   R¹⁰ is amido or a bond;    -   R¹¹ is H or alkyl;    -   W is a MetAP2 inhibitor moiety;    -   p is 0 to 20;    -   q is 2 or 3; and    -   r is 1, 2, 3, 4, 5, or 6.

In certain embodiments, Z is H. In other embodiments, Z is H₂N-AA₆-C(O)—

In certain embodiments, AA₆ is glycine.

In certain embodiments, Q is NR.

In certain embodiments, M is a bond.

In certain embodiments, J is a bond.

In certain embodiments, Y is NR.

In certain embodiments, W is:

wherein R₂ is —OH or methoxy; and R₃ is H, —OH or methoxy.

In certain embodiments, W is

In certain embodiments, W is

In certain embodiments, -Q-X—Y— is

V is

or a bond;

R¹² is H or Me; or R¹² taken together with R¹⁴ forms a piperidine ring;

R¹¹ is H or Me; and

R¹³ taken together with R¹² forms a piperidine ring.

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is;

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

Exemplary polymers of the invention have been described in U.S. Pat. No.4,997,878 to Bock et al, U.S. Pat. No. 5,037,883 to Kopecek et al. U.S.Pat. No. 5,258,453 to Kopecek et al, U.S. Pat. No. 6,464,850 to Zhang etal., U.S. Pat. No. 6,803,438 to Brocchini et al., each of which isincorporated by reference in its entirety. Additional exemplary polymershave been described in Subr et al., J Controlled Release, 18, 123-132(1992). Exemplary peptides of the invention have been described in U.S.Pat. No. 6,835,807 to Susaki et al, U.S. Pat. No. 6,291,671 to Inoue etal, U.S. Pat. No. 6,811,996 to Inoue et al, U.S. Pat. No. 7,041,818 toSusaki et al, U.S. Pat. No. 7,091,186 to Senter et al, U.S. Pat. No.7,553,816 to Senter et al. each of which is incorporated by reference inits entirety. Additional exemplary peptides and their cleavage have beendescribed in Shiose et al. Biol. Pharm. Bull. 30(12) 2365-2370 (2007)and Shiose et al. Bioconjugate Chem. 20(1) 60-70 (2009).

In some embodiments, the method of synthesis of the polymer may lead tothe coupling of two or more polymer chains and may increase the weightaverage molecular weight of the polymer conjugate. It is furtherrecognized that if this coupling occurs, the linkages will bebiodegradable.

Exemplary MetAP2 inhibitors have been described in U.S. Pat. No.6,242,494 to Craig et al, U.S. Pat. No. 6,063,812 to Hong et al, U.S.Pat. No. 6,887,863 to Craig et al, U.S. Pat. No. 7,030,262 to BaMaung etal., U.S. Pat. No. 7,491,718 to Comess et al, each of which isincorporated by reference in its entirety. Additional exemplary MetAP2inhibitors have been described in Wang et al. “Correlation of tumorgrowth suppression and methionine aminopeptidase-2 activity blockadeusing an orally active inhibitor,” PNAS 105(6) 1838-1843 (2008); Lee atal. “Design, Synthesis, and Antiangiogenic Effects of a Series of PotentNovel Fumagillin Analogues,” Chem. Pharm. Bull. 55(7) 1024-1029 (2007);Jeong et al. “Total synthesis and antiangiogenic activity ofcyclopentane analogues of fumagillol,” Bioorganic and MedicinalChemistry Letters 15, 3580-3583 (2005); Arico-Muendel et al. “CarbamateAnalogues of Fumagillin as Potent, Targeted Inhibitors of MethionineAminopeptidase-2,” J. Med. Chem. 52, 8047-8056 (2009); and InternationalPublication No. WO 2010/003475 to Heinrich et al.

Because the scientific literature has established a causal link betweeninhibition of MetAP2 and the resultant inhibition of endothelial cellproliferation and angiogenesis, it can be inferred that the MetAP2inhibitors described herein possess antiangiogenic activity. Asangiogenesis inhibitors, such compounds are useful in the treatment ofboth primary and metastatic solid tumors, including carcinomas ofbreast, colon, rectum, lung, oropharynx, hypopharynx, esophagus,stomach, pancreas, liver, gallbladder and bile ducts, small intestine,urinary tract (including kidney, bladder, and urothelium), femalegenital tract (including cervix, uterus, and ovaries as well aschoriocarcinoma and gestational trophoblastic disease), male genitaltract (including prostate, seminal vesicles, testes, and germ celltumors), endocrine glands (including the thyroid, adrenal, and pituitaryglands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma) and tumors of the brain, nerves, eyes, and meninges (includingastrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas, and meningiomas). Such compounds may alsobe useful in treating solid tumors arising from hematopoieticmalignancies such as leukemias (i.e., chloromas, plasmacytomas and theplaques and tumors of mycosis fungosides and cutaneous T-celllymphoma/leukemia) as well as in the treatment of lymphomas (bothHodgkin's and non-Hodgkin's lymphomas). In addition, these compounds maybe useful in the prevention of metastases from the tumors describedabove either when used alone or in combination with radiotherapy and/orother chemotherapeutic agents. The compounds of the invention can alsobe useful in the treatment of the aforementioned conditions bymechanisms other than the inhibition of angiogenesis.

Further uses include the treatment and prophylaxis of diseases such asblood vessel diseases such as hemagiomas, and capillary proliferationwithin atherosclerotic plaques; Osier-Webber Syndrome; myocardialangiogenesis; plaque neovascularization; telangiectasia; hemophiliacjoints; angiofibroma; and wound granulation. Other uses include thetreatment of diseases characterized by excessive or abnormalproliferation of endothelial cells, including not limited to intestinaladhesions, Crohn's disease, atherosclerosis, scleroderma, andhypertrophic scars, i.e., keloids. Another use is as a birth controlagent, by inhibiting ovulation and establishment of the placenta. Thecompounds of the invention are also useful in the treatment of diseasesthat have angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minutesalia quintosa) and ulcers (Helicobacter pylori).The compounds of the invention are also useful to reduce bleeding byadministration prior to surgery, especially for the treatment ofresectable tumors.

Another aspect of the present invention relates to a pharmaceuticalcomposition, comprising any one of the compounds described herein, and apharmaceutically acceptable carrier or excipient. In certainembodiments, the pharmaceutical composition comprises DMSO.

Yet another aspect of the present invention relates to a method oftreating a disease or condition by administering to a subject in needthereof a therapeutically effective amount of a compound or compositiondescribed herein, wherein the disease is cancer, a disease characterizedby irregular vasculature, a disease or condition characterized byhyperpermeable vasculature, cardiovascular, coronary vasculitis, pleuraleffusion, IL-2 associated edema, edema, or transplant rejection. Incertain embodiments, the disease is a solid tumor. In certainembodiments, the solid tumor is a melanoma, metastases, adenocarcinoma,sarcoma, thymoma, lymphoma, lung tumor, liver tumor, colon tumor, kidneytumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterinetumor, breast tumor, testicular tumor, bone tumor, muscle tumor, tumorof the head and neck, esophagus tumor, thyroid tumor, nasopharyngealtumor, endocrine tumor, brain tumor, tumor of the skin, soft tissuetumor, tumor of the placenta or gastric tumor.

Another aspect of the present invention relates to a method of treatingan angiogenic disease by administering to a subject in need thereof atherapeutically effective amount of a compound or composition describedherein.

Another aspect of the present invention relates to a method of treatingcancer by administering to a subject in need thereof a therapeuticallyeffective amount of a compound or composition described herein. Incertain embodiments, the cancer is adenocarcinoma, anal, astrocytoma,bladder, blood, bone, brain, breast, carcinoma, colon, cervical,endocrine, endometrial, esophageal, eye, gastric, genital, head andneck, hemangioma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, kidney,laryngeal, leukemia, liver, lung, lymphoma, melanoma, mesothelioma,metastatic, mouth, muscle, myeloma, nasal, nasopharyngeal, oral,ovarian, pancreatic, penile, placenta, prostate, rectal, renal, sarcoma,skin, soft tissue, testicular, throat, thymoma, thyroid, transitionalcell, ureter, uterine or vaginal.

Another aspect of the present invention relates to a method of treatmentor inhibition of an undesirable proliferation of cells by administeringto a subject in need thereof a therapeutically effective amount of acompound or composition described herein.

The compounds of the present invention are useful in inhibiting theproliferation of endothelial cells, tumor cells, smooth muscle cells,metastatic cells and others both in vitro and in vivo. Of particularinterest is the prevention or inhibition of endothelial celldifferentiation into capillary structures. The endothelial cellsamenable to inhibition by the compounds of the invention are present atseveral sites in a mammal and include but are not limited to dermis,epidermis, endometrium, retina, surgical sites, gastrointestinal tract,liver, kidney, reproductive system, skin, bone, muscle, endocrinesystem, brain, lymphoid system, central nervous system, respiratorysystem, umbilical cord, breast tissue, urinary tract and the like. Themethods of treatment of the present invention using the compoundsdescribed herein are particularly useful in preventing or inhibitingendothelial cell proliferation at sites of irregular vasculature,hyperpermeable vasculature, inflammation and tumorigenesis.

The compounds of the invention are particularly useful in methods ofinhibiting tumorigenesis in a mammal. Tumors which may be prevented orinhibited by preventing or inhibiting tumor cell proliferation with thecompound include but are not limited to melanoma, metastases,adenocarcinoma, sarcomas, thymoma, lymphoma, lung tumors, liver tumors,colon tumors, kidney tumors, non-Hodgkins lymphoma, Hodgkins lymphoma,leukemias, multiple myeloma, uterine tumors, breast tumors, prostatetumors, renal tumors, ovarian tumors, pancreatic tumors, brain tumors,testicular tumors, bone tumors, muscle tumors, tumors of the placenta,gastric tumors and the like.

In certain embodiments, the subject is a vertebrate. In certainembodiments, the vertebrate is a mammal. In certain embodiments, themammal is a human.

In providing a mammal with one or more of the compounds describedherein, the dosage of administered compound(s) will vary depending uponsuch factors as the mammal's age, weight, height, sex, general medicalcondition, previous medical history, disease progression, tumor burden,route of administration, formulation and the like. For example, asuitable dose of a compound of the invention for a mammal in need oftreatment as described herein is in the range of about 0.01 mg to about2000 mg compound per kilogram of body weight. In addition, due to theeffects of being bound to the polymer, agents may be administered atlower doses than typically used in the treatment of a particulardisorder. Surprisingly, in some embodiments the polymer conjugates ofthe invention are more active on a weight/weight basis than thecorresponding small molecules.

The present invention also encompasses combination therapy in whichcompounds described herein are used in combination with, for example, achemotherapeutic agent, or an anti-hypertensive agent. The therapeuticagents may also be conjugated to a polymer.

The route of administration may be intravenous (I.V.), intramuscular(I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (LP.),intrathecal (I.T.), intrapleural, intrauterine, rectal, vaginal,topical, intratumor and the like.

DEFINITIONS

The term “alkyl” refers to a fully saturated branched or unbranchedcarbon chain radical having the number of carbon atoms specified, or upto 30 carbon atoms if no specification is made. For example, a “loweralkyl” refers to an alkyl having from 1 to 10 carbon atoms, such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, andthose which are positional isomers of these alkyls. Alkyl of 10 to 30carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, certain cycloalkylshave from 3-10 carbon atoms in their ring structure, and may have 5, 6,or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl”, asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, or from one to six carbon atoms in its backbonestructure such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Throughout the application, certain alkylgroups are lower alkyls. In certain embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

“Alkenyl” refers to any branched or unbranched unsaturated carbon chainradical having the number of carbon atoms specified, or up to 26 carbonatoms if no limitation on the number of carbon atoms is specified; andhaving 1 or more double bonds in the radical. Alkenyl of 6 to 26 carbonatoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,where the unsaturated bond(s) can be located anywhere in the radical andcan have either the (Z) or the (E) configuration about the doublebond(s).

The term “alkynyl” refers to hydrocarbyl radicals of the scope ofalkenyl, but having one or more triple bonds in the radical.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O— alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R₁, where m and R₁ are described below.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be poly cycles. Heterocyclyl groups include, for example,thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,sulfamoyl, sulfmyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester,a heterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH₂)_(m)—R₁, wherein m and R₁ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates F, Cl, Br or I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formulae:

wherein R₃, R₅ and R₆ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R₁, or R₃ and R₅ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R₁ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In certain embodiments, only one of R₃or R₅ can be a carbonyl, e.g., R₃, R₅ and the nitrogen together do notform an imide. In certain embodiments, R₃ and R₅ (and optionally R₆)each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₁. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₃ and R₅ is an alkyl group. Incertain embodiments, an amino group or an alkylamine is basic, meaningit has a pK_(a)≧7.00. The protonated forms of these functional groupshave pK_(a)s relative to water above 7.00.

The term “carbonyl” (C(O)) is art-recognized and includes such moietiesas can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₇represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₁ or apharmaceutically acceptable salt, R₈ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R₁, where m and R₁ are as defined above. Where Xis an oxygen and R₇ or R₈ is not hydrogen, the formula represents an“ester”. Where X is an oxygen, and R₇ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₇ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R₈ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₇ or R₈ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R₇ is hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R₈ ishydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R₇ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R₇ is hydrogen, theabove formula represents an “aldehyde” group.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that “substitution” or “substitutedwith” includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₃ and R₅ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is as defined above.

The term “sulfamido” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₂ and R₄ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfmyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₁₂ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals, substantiallynon-pyrogenic, without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, not injurious to thepatient, and substantially non-pyrogenic. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include: (1)sugars, such as lactose, glucose, and sucrose; (2) starches, such ascorn starch and potato starch; (3) cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter, DMSO and suppository waxes; (9) oils,such as peanut oil, cottonseed oil, safflower oil, sesame oil, oliveoil, corn oil, and soybean oil; (10) glycols, such as propylene glycol;(11) polyols, such as glycerin, sorbitol, mannitol, and polyethyleneglycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar;(14) buffering agents, such as magnesium hydroxide and aluminumhydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonicsaline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphatebuffer solutions; and (21) other non-toxic compatible substancesemployed in pharmaceutical formulations. In certain embodiments,pharmaceutical compositions of the present invention are non-pyrogenic,i.e., do not induce significant temperature elevations when administeredto a patient.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts, and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

In other cases, the compounds useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

A “therapeutically effective amount” of a compound, with respect to usein treatment, refers to an amount of a compound in a preparation which,when administered as part of a desired dosage regimen (to a mammal,preferably a human) alleviates a symptom, ameliorates a condition, orslows or prevents the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment. A “therapeutically effectiveamount” is synonymous with “efficacious dose”.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human subject.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “amino acid” is intended to embrace all compounds, whethernatural or synthetic, which include both an amino functionality and anacid functionality, including amino acid analogs and derivatives. Incertain embodiments, the amino acids contemplated in the presentinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids, which contain amino and carboxyl groups. Naturally occurringamino acids are identified throughout by the conventional three-letterand/or one-letter abbreviations, corresponding to the trivial name ofthe amino acid, in accordance with the following list. The abbreviationsare accepted in the peptide art and are recommended by the IUPAC-IUBcommission in biochemical nomenclature.

By the term “amino acid residue” is meant an amino acid. In general theabbreviations used herein for designating the naturally occurring aminoacids are based on recommendations of the IUPAC-IUB Commission onBiochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). Forinstance Met, He, Leu, Ala and Gly represent “residues” of methionine,isoleucine, leucine, alanine and glycine, respectively. By the residueis meant a radical derived from the corresponding a-amino acid byeliminating the OH portion of the carboxyl group and the H portion ofthe a-amino group.

The term “amino acid side chain” is that part of an amino acid residueexclusive of the backbone, as defined by K. D. Kopple, “Peptides andAmino Acids”, W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H—(the side chain of glycine). These side chains are pendant from thebackbone Cα carbon.

The term “peptide,” as used herein, refers to a sequence of amino acidresidues linked together by peptide bonds or by modified peptide bonds.The term “peptide” is intended to encompass peptide analogs, peptidederivatives, peptidomimetics and peptide variants. The term “peptide” isunderstood to include peptides of any length. Peptide sequences set outherein are written according to the generally accepted conventionwhereby the N-terminal amino acid is on the left, and the C-terminalamino acid is on the right (e.g., H₂N-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-CO₂H).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomer.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

General Procedures

Tangential Flow Filtration (TFF) was used to purify the polymer productsof the invention. TFF was performed with a Pall Minimate™ Capsule andMinimate™ TFF system according to the manufacturer's instructions.Either a Minimate TFF Capsule with 5 kDa Omega membrane (5K) or MinimateTFF Capsule with 10 kDa Omega membrane (10K) cartridge was used forpurification. In all cases, the permeate was discarded and the retentatelyophilized to yield the polymer product. Structures of products wereconfirmed by ¹H NMR, small molecules were also characterized by MS.Polymer weights reported in the examples were not corrected for watercontent.

Carbamoylfumagillol and chloroacetylcarbamoylfumagillol can be preparedaccording to the methods disclosed in U.S. Pat. No. 5,166,172(Kishimoto, et al., incorporated herein by reference). p-Nitrophenylfumagill-6-yl carbonate can be prepared according to publishedprocedures. (See Han, C. et al. Biorg. Med. Chem. Lett. 2000, 10,39-43). MA-GFLG-ONp can be prepared according to the methods disclosedin U.S. Pat. No. 5,258,453 (Kopecek et al. incorporated herein byreference.)

Example 1 Synthesis of poly(HPMA-co-MA-GFLG-ONp)

A mixture of hydroxypropylmethacrylamide (HPMA, 22.16 g, 155 mmol),N-methyacryl-gly-phe-leu-gly p-nitrophenyl ester (MA-GFLG-ONp, 10.00 g,17.19 mmol), AIBN (1.484 g, 9.037 mmol) and acetone (225 g) was degassed(freeze, pump, thaw, 4 cycles). The resulting reaction mixture wasstirred at 50° C. for 48 hours, then cooled to room temperature. Thedesired product was purified by trituration with acetone, then driedunder vacuum to yield 17.6 g of poly(HPMA-co-MA-GFLG-ONp) as a whitesolid. The structure was verified by ¹H NMR and the product shown to befree from substantial impurities (e.g., p-nitrophenol). Based on UVabsorbance, the copolymer contained 0.47 mmoles of p-nitrophenyl esterper gram of polymer. The copolymer of this example is used in most ofthe subsequent examples. A wide range of copolymers based on differentmonomers and/or monomer ratios may be made following this procedure byadjusting the stoichiometry and/or using different monomers.

Example 2 Synthesis of poly(HPMA-co-MA-GFLG-OH)

Poly(HPMA-co-MA-GFLG-ONp) (700 mg) was added portionwise to a solutionof 0.1 M NaOH (11.3 mL) at 0° C. The yellow reaction mixture was stirredat 0° C. for 0.5 hours, then at room temperature for 4 hours. One-halfof the solution was acidified with 0.1 M HCl to pH=6. The aqueous phasewas extracted with ethyl acetate to remove excess p-nitrophenol. Theaqueous phase was lyophilized to afford poly(HPMA-co-MA-GFLG-OH) as acolorless solid (360 mg).

Example 3 Synthesis of poly(HPMA-co-MA-GFLG-NHCH2CH2N(Me)BOC) andGeneral Procedure A

A solution of poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6mL) and H₂O (10 mL) was added dropwise over a 15 minute interval to asolution of tert-butyl N-(2-aminoethyl)-N-methylcarbamate (0.20 g, 1.15mmol) in water (20 mL) at 0° C. The reaction mixture was stirred at 0°C. for 15 minutes, then warmed to room temperature and stirred for 12hours. The solvents were evaporated under reduced pressure. Theresulting residue was dissolved in water (50 mL), the pH was adjusted toapproximately 8.0 with 0.1 M NaOH. The solution was filtered through aVacuCap filter, then purified using TFF (10 K). The polymer-containingsolution was washed (as part of the TFF process) with 25 mM NaClsolution (800 mL) to remove p-nitrophenol, the pH of the solution wasadjusted to approximately 4 with 0.1 M HCl, and then washed (as part ofthe TFF process) with water (400 mL). The polymer solution waslyophilized to isolate the compoundpoly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)BOC) as a pale yellow solid (720 mg,71%).

Example 4 Synthesis of poly(HPMA-co-MA-GFL G-NHCH₂CH₂NHMe)

A solution of poly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)BOC) (260 mg, 0.136mmol) in D₂O (5.2 mL) was irradiated with microwave radiation at 150° C.with stirring for 6 hours. The ¹H NMR of this material indicated thatdeprotection of BOC group had occurred. The aqueous solution waslyophilized to isolate the poly(HPMA-co-MA-GFLG-NHCH₂CH₂NHMe) as a paleyellow solid (210 mg, 85%).

Example 5 Synthesis ofN-({[2-(acetylamino)ethyl](methyl)amino}acetyl)carbamoylfumagillol andGeneral Procedure B

Diisopropylethylamine (DIEA) (130 mg) was added to a solution ofN-[2-(methylamino)ethyl]acetamide hydrochloride (76 mg) andchloroacetylcarbamoylfumagillol (200 mg) in anhydrous DMF at 0° C. underN₂. The reaction mixture was allowed to warm to room temperature, andstirred for 12 hours. The solvent was removed under reduced pressure andthe resulting residue was suspended in water (30 mL) and extracted withEtOAc (aqueous and organic phases from the emulsion formed wereseparated using a centrifuge) to remove excesschloroacetylcarbamoylfumagillol. Nitrogen was passed through the aqueoussolution to reduce the residual level of EtOAc. The product was purifiedby flash chromatography (methanol/methylene chloride) to yieldN-({[2-(acetylamino)ethyl](methyl)amino}acetyl)carbamoylfumagillol (75mg) as an off-white foam.

Example 6 BocNHCH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl (Alkylation ofN—BOC, N′-methylethylenediamine with chloroacetylcarbamoylfumagillol)

A solution of TNP-470 (0.2 g) and DIEA (0.105 g) in DMF (3 mL) wascooled to 0° C. A solution of tert-butylN-[2-(methylamino)ethyl]carbamate (0.105 g) in DMF (3 mL) was added, andthe mixture was stirred for 3 hours at 0° C. and then overnight. Thereaction was diluted with ethyl acetate and extracted with water. Theaqueous phase was back extracted with ethyl acetate, and the combinedorganic phases were extracted with brine, dried (MgSO₄) and evaporatedto afford an oil. Purification by silica gel chromatography(methanol/methylene chloride) and evaporation of the product fractionsgave BocNHCH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl a white foam (0.16 g,60%).

Example 7 Reaction of tert-butyl N-[2-aminoethyl] carbamate withchloroacetylcarbamoylfumagillol

A 30 uL aliquot of a 1 M solution of Boc-ethylenediamine in DMF wasadded to DMF (270 uL). The solution was cooled to 0° C., and a solutionof TNP-470 (48 mg) in DMF (600 uL) was added dropwise over 2 minutes.The reaction was monitored by LC/MS. The largest amount of the desiredalkylation product observed was 34%. Carbamoylfumagillol was alsoproduced. The ratio of desired product to carbamoylfumagillol was 1.0 to0.4. Attempted isolation of the desired product resulted in theisolation of hydantoin and fumagillol.

Example 8 Synthesispoly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)CH2C(O)NHC(O)₂-fumagill-6-yl)

General Procedure B was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂NHMe) (105 mg, 0.058 mmol) andchloroacetylcarbamoylfumagillol (46 mg, 0.114 mmol) in DMF (5 mL) towhich DIEA (29.5 mg, 0.228 mmol) was added N₂. The product was purifiedusing TFF (5 K) by washing with water (150 mL) to remove DIEAhydrochloride. The polymer solution was lyophilized to obtain thepolymer conjugate (60 mg, 48%) as a pale yellow solid.

Example 9 Synthesis of pol(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂HCl) and GeneralProcedure C for the reaction of diamines with poly(HPMA-co-MA-GFLG-ONp)

A solution of ethylenediamine (0.33 g, 5.49 mmole) in water (20 mL), pH11.7, was adjusted to pH 9.1 by the addition of 37% aq HCl (17-18drops). The solution was cooled in an ice bath andpoly(HPMA-co-MA-GFLG-ONp) (1.03 g) in DMF (6 mL) was added dropwise over20 minutes while maintaining the temperature below 4° C. The solutionwas stirred 20 minutes at 4° C., 50 minutes at room temperature to givea lemon yellow solution, pH 8.1. The solution was evaporated at 40° C.H₂O (3×10 mL) was added and evaporated. The product was diluted withwater (60 mL), the solution adjusted with NaOH to pH 8.0. The solutionwas filtered through a VacuCap filter and purified by TFF as follows.The polymer solution was first washed with 25 mM NaCl solution (800 mL)to remove p-nitrophenol. The solution was washed with water (400 mL)then adjusted to pH 4 with 0.1 M HCl. The TFF retentate was collectedand the filter was washed with 2×10 mL of water. The combined retentateand washes gave a polymer solution which was lyophilized to isolate thecompound poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂ HCl) as a pale yellow solid(0.71 g, 72%).

Example 10 Synthesis of poly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂NHMe-HCl)

General Procedure C was followed using N,N′-dimethylethylenediamine(0.47 g, 5.36 mmol) and poly(HPMA-co-MA-GFLG-ONp) (1.0 g) to yieldpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂NHMe HCl) as an off-white solid (0.78g).

Example 11 Synthesis ofpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl)

General procedure B was followed usingpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂NHMe) (200 mg, 0.108 mmol) andchloroacetylcarbamoylfumagillol (86 mg, 0.213 mmol) to yieldpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl) as apale yellow solid (180 mg).

Example 12 Synthesis of N-f (2R) l-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol and General Procedure D

A solution of p-nitrophenyl fumagill-6-yl carbonate (400 mg, 0.89 mmol)and (R)-2-amino-3-methyl-1-butanol (280 mg, 2.71 mmol) were stirred inethanol (10 mL) at room temperature for 12 hours. The yellow solutionwas concentrated and the residue purified by flash chromatography(methanol/methylene chloride) to yieldN-[(2R)l-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol (340 mg, 0.83mmol) as a colorless oil.

Example 13 Synthesis of N-(6-hydroxyhexyl)carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (150 mg) in ethanol (10 mL) and 6-aminohexanol (48 mg). Theproduct was isolated as a colorless oil (110 mg, 78%).

Example 14 Synthesis ofN-[l-(hydroxymethyl)cyclopentyl]carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (100 mg) in ethanol (3 mL) and THF (1 mL) and cycloleucinol(52 mg) to afford N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol asan oil (50 mg).

Example 15 Synthesis ofN-(l-hydroxy-2-methylpropan-2-yl)carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (100 mg) in ethanol (3 mL) and THF (2 mL) and2-amino-2-methylpropanol (40 mg) to affordN-(1-hydroxy-2-methylpropan-2-yl)carbamoylfumagillol as an oil (37 mg).

Example 16 Synthesis of fumagill-6-yl(2S)-2-(hydroxymethyl)pyrrolidine-l-carboxylate

General procedure D was followed. The S-prolinol (68 mg, 0.67 mmol) wasreacted with p-nitrophenyl fumagill-6-yl carbonate (150 mg, 0.335 mmol)in ethanol (4 mL) The product was purified by flash chromatography(methanol/methylene chloride) to yield fumagill-6-yl(2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate as a white foam (81 mg,63%).

Example 17 Synthesis of fumagill-6-yl(2S)-2-({[(chloroacetyl)carbamoyl]oxy}methyl)pyrrolidine-l-carboxylate

A solution of fumagill-6-yl(2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (330 mg) in methylenechloride (2.1 mL) was cooled to 0° C. and chloroacetylisocyanate (115mg) in methylene chloride (1.5 mL) was added dropwise. After 40 minutes,the mixture was diluted with methylene chloride (20 mL) and the organicphase washed with water (3×). The organic phase was dried (Na₂SO₄) andevaporated to yield fumagill-6-yl(2S)-2-({[(chloroacetyl)carbamoyl]oxy}methyl)pyrrolidine-1-carboxylateas a white foam (400 mg).

Example 18 Synthesis ofpoly[HPMA-co-MA-GFLG-NCH₂CH₂N(Me)-acetylcarbamoyl-[(2R)-1-hydroxy-3-methylbutan-2-yl]carbamoylfumagillol]

General procedure B was followed usingchloroacetylcarbamoyl[(2R)-1-hydroxy-3-methylbutan-2-yl]carbamoylfumagillol(120 mg) (and poly(HPMA-co-MA-GFLG-NHCH₂CH₂NHMe) (200 mg) with DIEA (57mg) in DMF (5 mL) to yield2-poly[HPMA-co-MA-GFLG-NCH₂CH₂N(Me)]-acetylcarbamoyl-[1-hydroxy-3-methylbutan-2-yl]carbamoylfumagillol(200 mg, 80%).

Example 19 Synthesis of fumagill-6-yl2-(poly[HPMA-co-MA-GFLG-NCH₂CH₂N(Me)]-acetylcarbamoylhydroxymethyl)pyrrolidine-l-carboxylate)

General procedure B was followed using the fumagill-6-yl(2S)-2-(chloroacetylcarbamoylhydroxymethyl)pyrrolidine-1-carboxylate (90mg) (and poly(HPMA-co-MA-GFLG-NHCH₂CH₂NHMe) (200 mg) with DIEA (57 mg)in DMF (5 mL) to yield fumagill-6-yl 2-poly[HPMA-co-MA-GFLG-NCH₂CH₂N(Me)]-acetylcarbamoylhydroxymethyl)pyrrolidine-1-carboxylateas a pale yellow solid (150 mg, 60%).

Example 20 Synthesis of N-(6-aminohexyl)carbamoylfumagillol

A solution of 1,6-diaminohexane (0.13 g) in methanol (8 mL) was cooledto 0° C. and p-nitrophenyl fumagill-6-yl carbonate (0.13 g) in methanol(2 mL) was added dropwise. The solvent was reduced to about 2 mL byrotary evaporation. Ethyl acetate was added and the organic phase waswashed with water, 0.1 N NaOH, water, brine and dried with sodiumsulfate. The solvent was evaporated and the residue dissolved in ethanol(15 mL). DL-tartaric acid (16 mg) was added, the solution was storedovernight and then evaporated to about 0.5 mL. Ether was added and awhite solid formed. The solid was collected by filtration, washed withether and dried to yield the tartrate salt ofN-(6-aminohexyl)carbamoylfumagillol (74 mg).

Example 21 Synthesis of poly [HPMA-co-MA-GFLG-NH(CH₂)₆NH₂HCl]

General Procedure C was followed using 1,6-diaminohexane (621 mg, 5.36mmol) and poly(HPMA-co-MA-GFLG-ONp) (1.0 g). The crude product waspurified by TFF (5 K) using aqueous NaCl (25 mM) and then acidified topH 4.0 with 0.1 M HCl and further purified by TFF with water to yieldpoly[HPMA-co-MA-GFLG-NH(CH₂)₆NH₂ HCl] as an off-white solid (860 mg).

Example 22 Synthesis of p-nitrophenyl N-[(2R)l-hydroxy-2-methylbutan-2-yl]carbamoylfumagill-6-yl carbonate andGeneral Procedure E

To a solution of the alcoholN-[(2R)l-hydroxy-2-methylbutan-2-yljcarbamoylfumagillol (1.11 g) inmethylene chloride at 0° C. under N₂ was added DMAP (660 mg, 5.40 mmol)followed by the portionwise addition of p-nitrophenyl chloro formate(810 mg). The reaction mixture was stirred at 0° C. for 1 hour. Thesolvent was evaporated and the resulting residue was dissolved in EtOAcand washed with water, brine and dried (Na₂SO₄). Evaporation of EtOAcprovided the crude product, which was purified by flash chromatography(silica, eluting with 100% hexanes and then with 2-30% EtOAc). Thefractions containing pure product were combined and evaporated toisolateN-[(2R)1-(p-nitrophenolcarbonylhydroxy-2-methylbutan-2-yl]carbamoylfumagillol(1.25 g, 80%) as a white solid.

Example 23 Synthesis ofN-[1-(p-nitrophenoxycarbonylhydroxymethyl)-2-methylpropan-2-yl)carbamoylfumagillol

Following General Procedure E, dimethylalcohol (60 mg), p-nitrophenylfumagill-6-yl carbonate (46 mg), and DMAP (37 mg) were reacted inmethylene chloride (8 mL). The reaction mixture was diluted with ethylacetate and washed with water (3×) and then brine. The organic phase wasdried (Na₂SO₄) and evaporated to a yellow foam (87 mg) which was usedwithout further purification.

Example 24 Synthesis ofN-[1-(p-nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol

Following General Procedure E,N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol (product fromExample 14, 74 mg), p-nitrophenyl chloroformate (53 mg), and DMAP (43mg) were reacted in methylene chloride (5 mL). After the extractiveworkup,N-[1-(p-nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol(100 mg) was used without further purification.

Example 25 Synthesis ofpoly[HPMA-co-MA-GFLG-NH(CH₂)₆NHcarbamoyl-[1-hydroxy-3-methylbutan-2-yl]carbamoylfumagillol]and General Procedure F

To a solution of polymer (400 mg) and p-nitrophenylN-[(2R)l-hydroxy-3-methylbutan-2-yl]carbamoylfumagill-6-yl carbonate(240 mg) in DMF (8 mL) at 0° C. was added DIEA (0.11 g) dropwise. Thesolution was stirred at 0° C. for one hour and allowed to warm to roomtemperature. After 3 days, the solvent was evaporated and water (80 mL)was added. The aqueous phase was extracted with ethyl acetate (500 mLtotal) until none of the starting carbonate was detectable by MS. Theaqueous phase was purified by TFF (10 K) and the retentate lyophilizedto yield the conjugate as a white solid (380 mg, 77%).

¹H NMR (DMSO-d6): δ 8.25 (bs, 2H, amide-NH), 8.0 (bs, 1H, amide-NH),7.70 (bs, 2H, amide-NH), 7.10-7.30 (m, 15H, Phenylalanine and amide-NH),7.10 (bt, 1H, NH-Fum), 6.92 (bd, 1H, NH-Fum), 5.26 (m, H-5-Fum), 5.18(bt, alkene-Fum), 4.50-4.80 (m, 1H, phenylalanine alpha proton),4.0-4.21 (m, 1H, leucine alpha proton), 3.50-3.84 (m, 19H), 3.29 (s, 3H,OMe-Fum), 2.80-3.10 (m, 28H), 2.51 (d, 1H, J=4.4 Hz, H-2-Fum), 2.19 (m,2H, allylic-Fum), 0.82-1.92 [m, 131H {1.84 (m, 2H, Fum), 1.72 (s, 3H,Fum-Me), 1.60 (s, 3H, Fum-Me), 1.09 (s, 3H, Fum-Me), 0.84 (dd, 6H,Fum-isopropyl}].

Example 26 Synthesis ofpoly[HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoylfumagillol]

General procedure F was followed using poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂HCl) (200 mg), p-nitrophenyl fumagill-6-yl carbonate (100 mg) and DIEA(57 mg) in DMF (10 mL). The product was purified by TFF (10 K) withwater and lyophilized to yield the conjugate as a pale yellow solid (160mg).

Example 27 Synthesis ofpoly[HPMA-co-MA-GFLG-N(Me)-(2-methylaminoethyl)carbamoylfumagillol]

General procedure F was followed usingpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂NHMe HCl) (200 mg), p-nitrophenylfumagill-6-yl carbonate (100 mg) and DIEA (57 mg) in DMF (5 mL). Theproduct was purified using TFF (10 K) with water and lyophilized toyield the conjugate as an off-white solid (180 mg).

Example 28 Synthesis ofpoly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagillol

General procedure F was followed using poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂HCl) (200 mg), p-nitrophenyl dihydrofumagill-6-yl carbonate (200 mg) andDIEA (57 mg) in DMF (10 mL). The product was purified by TFF (10 K) withwater (150 mL) and lyophilized to yieldpoly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagillol as apale yellow solid (160 mg).

Example 29 Synthesis ofpoly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol]

General procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂CH₂NH₂ HCl) (220 mg), p-nitrophenylfumagill-6-yl carbonate (110 mg) and DIEA (63 mg) in DMF (6 mL). Thesolvent was evaporated and the resulting solution diluted with water.The aqueous phase was extracted with ethyl acetate and purified by TFFusing 350 mL of water. The retentate was lyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol] as a lightpink powder (200 mg).

Example 30 Synthesis ofpoly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol]

General procedure F was followed usingpoly[HPMA-co-MA-GFLG-N-(6-aminohexylamine.HCl)] (1.0 g), p-nitrophenylfumagill-6-yl carbonate (0.48 g) and DIEA (0.27 g) in DMF (25 mL). Thesolvent was evaporated and the solution diluted with water. The aqueousphase (300 mL) was extracted with ethyl acetate (700 mL total) andpurified by TFF using an additional 350 mL of water. The retentate waslyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol] as a lightpink solid (0.9 g).

¹H NMR (DMSO-d6): δ 8.10-8.35 (m, 3H, amide-NH), 7.90-8.10 (m,amide-NH), 7.05-7.32 (m, 22H, amide-NH) 5.27 (m, H-5-Fum), 5.18 (bt,alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, phenylalanine alphaproton), 4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H),3.27 (s, 3H, OMe-Fum), 2.80-3.20 (m, 33H), 2.56 (d, 1H, H=3.90 Hz,H-2-Fum), 2.18 (m, 2H, allylic-Fum), 0.37-2.0 [m, 147H {1.70 (s, 3H,Fum-Me), 1.60 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum-Me)}].

Example 31 Synthesis ofpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexyl)carbamoylfumagillol]

General procedure F was followed usingpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexylamine.HCl)] (1.0 g),p-nitrophenyl fumagill-6-yl carbonate (0.48 g) and DIEA (0.27 g) in DMF25 mL. The solvent was evaporated and the solution diluted with water.The aqueous phase (300 mL) was extracted with ethyl acetate (700 mLtotal) and purified by TFF using an additional 350 mL of water. Theretentate was lyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexyl)carbamoylfumagillol] asa light pink solid (0.9 g). ¹H NMR (DMSO-d6): δ 7.90-8.35 (m, 4H,amide-NH), 7.0-7.70 (m, 25H, Phenylalanine and amide-NH), 5.26 (m,H-5-Fum), 5.18 (bt, alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H,phenylalanine alpha proton), 4.10-4.30 (m, 1H, leucine alpha proton),3.40-3.80 (m, 21H), 3.26 (s, 3H, OMe-Fum), 2.80-3.10 (m, 31H), 2.17 (m,2H, allylic-Fum), 0.37-2.0 [m, 166H {1.69 (s, 3H, Fum-Me), 1.59 (s, 3H,Fum-Me), 1.07 (s, 3H, Fum-Me)}]

Example 32 Synthesis ofpoly[HPMA-co-MA-GFLG-N-[2-(4-aminophenyl)ethyl]carbamoylfumagillol]

To a suspension of poly[HPMA-co-MA-GFLG-OH] (200 mg),N-[2-(4-aminophenyl)ethyl]carbamoylfumagillol] (100 mg) and DIEA (75 mg)in DMF (6 mL) at 0° C. was added EDCI (total 44 mg) in portions. Thesolution was allowed to warm to room temperature and stirred overnight.The solvent was evaporated, the residue was suspended in water and thesuspension extracted with EtOAc (7 times, total 250 mL). The aqueousphase was purified by TFF (10 K) using water (350 mL). The retentate waslyophilized to afford the polymer as a white fluffy solid (170 mg).

Example 33 Synthesis ofpoly[HPMA-co-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoylfumagillol]

To a solution of 2,2′-(Ethylenedioxy)bis(ethylamine) (0.79 g, 5.34 mmol)in distilled water (20 mL) at 0° C. (pH=11.56) was added cone. HCl untilpH of the solution was 9.01 (measured by pH meter).Poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 mL) and H₂O (10mL) was added to the amine-containing solution dropwise over a period of15 minutes and the reaction mixture was stirred at 0° C. for 15 minutes.The reaction mixture was then allowed to warm to room temperature andstirred for 2 hours. The pH of the solution was measured to be 8.15. Thereaction mixture was diluted with distilled water (300 mL) and filteredthrough a VacuCap filter, reaction flask was washed with water (100 mL).The polymer solution was concentrated to 40 mL by TFF (10 K) and waswashed with 25 mM NaCl (800 mL) to remove p-nitrophenol, the pH was thenadjusted to 4 with 0.1 M HCl and finally washed with water (400 mL). Thepure polymer solution was lyophilized to isolatepoly[HPMA-co-MA-GFLG-NH-2-[2-(2-aminoethoxy)ethoxy]ethylamine-HCl] as apink solid (800 mg, 78%).

To a mixture of p-nitrophenyl fumagill-6-yl carbonate (93 mg, 0.208mmol) andpoly[HPMA-co-MA-GFLG-N-2-[(2-(2-aminoethoxy)]ethoxy)ethylamine-HCl] (200mg, 0.104 mmol) in anhydrous DMF (5 mL) at 0° C. under N₂ was added DIEA(57 mg, 0.416 mmol). The reaction mixture was allowed to warm to roomtemperature and stirred for 12 hours. The solvent was removed underreduced pressure and the resulting residue was suspended in water (30mL) and extracted with EtOAc (aqueous and organic phases from theemulsion formed were separated using centrifuge) to remove excess ofp-nitrophenyl fumagill-6-yl carbonate and p-nitrophenol. Nitrogen waspassed through the aqueous solution to remove traces of EtOAc and it waspurified using TFF (5K) by washing it with water (150 mL) to remove DIEAhydrochloride. The polymer solution was lyophilized to obtain thedesired polymer conjugatepoly[HPMA-co-MA-GFLG-N-2-[2-(2-aminoethoxy)ethoxy ethyl]carbamoylfumagillol] (220 mg, 95%) as an off-white solid.

Example 34 Synthesis ofpoly[HPMA-co-MA-GFLG-NH-(6-aminodecyl)carbamoylfumagillolJ]

To a mixture of p-nitrophenyl fumagill-6-yl carbonate (300 mg, 0.67mmol) and poly[HPMA-co-MA-GFLG-N-10-[decylamine-HCl] (300 mg, 0.15 mmol;made in a similar manner to Example 33 except 1,10-diaminodecane wasused as the amine) in anhydrous DMF (6 mL) at 0° C. under N₂ was addedDIEA (83 mg, 0.64 mmol). The reaction mixture was allowed to warm toroom temperature and stirred for 12 hours. The solvent was removed underreduced pressure and the resulting residue was suspended in water (30mL) and extracted with EtOAc (aqueous and organic phases from theemulsion formed were separated using a centrifuge) to remove excess ofp-nitrophenyl fumagill-6-yl carbonate and p-nitrophenol. Nitrogen waspassed through the aqueous solution to remove traces of EtOAc. The crudeaqueous solution was purified using TFF (10K) by washing with water (150mL) to remove DIEA hydrochloride. The polymer solution was lyophilizedto obtain the desired polymer conjugatepoly[HPMA-co-MA-GFLG-NH-(10-aminodecyl)carbamoylfumagillol] (300 mg,87%) as an off-white solid.

Example 35 Synthesis of N-(2-acetamidoethyl)carbamoylfumagillol

To a solution of p-nitrophenyl fumagill-6-yl carbonate (200 mg) inethanol (5 mL) at 0° C. was added N-(2-aminoethyl)acetamide (0.132 mL).The solution was stirred at 0° C. for one hour and overnight at roomtemperature. The reaction was diluted with ethyl acetate, washed withwater. The aqueous phase was back extracted with ethyl acetate and thecombined organic phases dried (MgS)₄). The crude product was purified byflash chromatography. The product was a yellow solid (120 mg).

Example 36 Lysine Conjugate of Polymer and Met AP 2 Inhibitor Moiety

To a solution of p-nitrophenyl fumagill-6-yl carbonate (400 mg) andN-ε-Cbz-O-methyl-L-lysine hydrochloride in DMF (10 mL) at 0° C. wasadded DIEA (350 mg). The reaction was warmed to room temperature and thestirred overnight. The solution was diluted with ethyl acetate, washedwith 0.1 N NaOH (4×), water, and then brine. The organic phase was dried(Na₂SO₄), filtered and evaporated. The residue was purified by flashchromatography (silica; methanol/methylene chloride) to provide theN-ε-Cbz-O— methyl-lysine-carbonylfumagillol (550 mg).

To a solution of N-ε-Cbz-O-methyl-lysine-carbonylfumagillol (200 mg) inethyl acetate (10 mL) was added PtO2 monohydrate (20 mg) and thesolution hydrogenated at STP for 20 minutes. Reduction of the doublebond but not deprotection of the Cbz was verified by MS. The solutionwas filtered and evaporated. The residue was dissolved in methanol (10mL) and 10% Pd/C (20 mg) was added. The solution was hydrogenated underSTP for 5 minutes, and removal of the Cbz group confirmed by MS. Thesolution was filtered with celite, and evaporated to provideO-methyl-L-Lys-carbonyldihydrofumagillol as a colorless oil (0.15 g).

To a stirred solution of O-methyl-L-Lys-carbonyldihydrofumagillol (150mg, 0.32 mmol) in DMF (6 mL) was added poly(HPMA-co-MA-GFLG-ONp) (300mg) at 0° C. The resulting yellow solution was allowed to warm to roomtemperature overnight. The solvent was evaporated and the residuesuspended in water (30 mL). The suspension was extracted six times withethyl acetate (total ethyl acetate volume=150 mL). The aqueous phase waslyophilized to provide the polymer conjugate as a white solid (180 mg,63%).

Example 37 Aminothiophenol Conjugate of Polymer and Met AP 2 InhibitorMoiety

To a solution of chloroacetylcarbamoylfumagillol (500 mg) and4-aminothiophenol (180 mg) in DMF (10 mL) at 0° C. was added DIEA (193mg). The solution was stirred at 0° C. for 1.5 hours and then at roomtemperature overnight. The solution was diluted with water and extractedwith ethyl acetate. Purification by flash chromatography (MeOH/CH₂Cl₂)followed by a second chromatography (EtOAc/hexanes) gave4-aminophenylthioacetylcarbamoylfumagillol (460 mg).

To a solution of poly(HPMA-co-MA-GFLG-ONp) (200 mg) and4-aminophenylthioacetylcarbamoylfumagillol (100 mg) in DMF (5 mL) at 0°C. was added DIEA (106 mg). The solution was allowed to warm to roomtemperature and then heated to 50° C. and stirred overnight. The solventwas evaporated and the residue suspended in water. The suspension wasextracted with ethyl acetate (150 mL). The aqueous phase was lyophilizedto yield the polymer conjugate as a white solid (180 mg).

Example 38

To a solution of poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂ HCl) (200 mg) andN-(5-carboxypentyl)carbamoylfumagillol (96 mg) in DMF (6 mL) at 0° C.was added DIEA (104 mg) followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (42 mg).The solution was allowed to warm to RT and stirred overnight. Thesolvent was evaporated and the residue dissolved in water (50 mL) andextracted with ethyl acetate (200 mL). The aqueous phase was purified byTFF with water (450 mL). The retentate was lyophilized to yield thepolymer (200 mg) as a pale yellow solid.

Example 39

To a solution of poly[HPMA-co-MA-GFLG-N(CH₂)₆NH₂ HCl] (216 mg),2-carboxyethylcarbamoylfumagillol (91 mg) in DMF (8 mL) at 0° C. wasadded DIEA (118 mg) followed byN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (88 mg).The solution was allowed to warm to room temperature and stirredovernight. The solvent was evaporated and the residue dissolved in water(50 mL) and extracted with ethyl acetate (200 mL). The aqueous phase waspurified by TFF (10 K) with water (1 L). The retentate was lyophilizedto yield the polymer (170 mg) as a pale yellow solid.

Example 40

General Procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂CH₂NH₂ HCl) (220 mg) and carbonate (Example24, 100 mg) in DMF (6 mL) with DIEA (63 mg). The reaction was extractedwith ethyl acetate. Following TFF (10 K) purification with water, andlyophilization, the product was isolated as a light pink powder (140mg).

Example 41

General Procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂CH₂NH₂ HCl) (200 mg) and carbonate (Example23, 86 mg) in DMF (5 mL) with DIEA (57 mg). Extraction was performedwith ethyl acetate. Following TFF purification with water, andlyophilization, the product was isolated as a light pink powder (200mg).

Example 42 Synthesis of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide]

To a solution of aminohexylpolymer (600 mg) and p-nitrophenyl acetate(110 mg) in DMF (16 mL) at 0° C. was added DIEA dropwise. The solutionwas allowed to warm to room temperature and stirred overnight. Thesolvent was evaporated and the residue was dissolved in water (50 mL),filtered through a vacu-cap filter with an additional 25 mL of water.The pH was adjusted to 8.0 with 0.1 M NaOH and the solution concentratedto 50 mL (TFF). The retentate was washed with aqueous NaCl (25 mM, 450mL) until the permeate was almost colorless and then washed with water(400 mL) to a conductivity of 0.00 uS. The retentate was lyophilized toyield 0.59 g of a pink solid.

Example 43 Aqueous Stability of Carbamoylfumagillol

A stock solution of carbamoylfumagillol in DMSO was diluted in a 15 mLpolypropylene screw top tube with either 5 mL of 10 mM sodium acetatebuffer at either pH 4.0 or 5.3, or potassium phosphate buffer at pH 6.7or 8.0 at 37° C. The final concentration of carbamoylfumagillol in thebuffer solution was 5 μM. At the appropriate time points, a 50 μL samplewas withdrawn and diluted with three volumes of methanol containingpropranolol as an internal standard (one solution was made for theentire study). The concentration of carbamoylfumagillol in the solutionwas analyzed by LC/MS/MS over seven days. From pH 5.3 to 8.0, less than20% decomposition was observed over the seven day period. Estimated rateconstants are presented in Table 1.

TABLE 1 Natural Rate Constant of Carbamoylfumagillol after Incubation37° C. in Aqueous Buffer at Various pHs pH Natural Rate Constant (hr⁻¹)T½ (hr) 4.0 0.0054 129 5.3 0.0017 407 6.7 0.0010 728 8.0 0.0011 613 *thevalues in italics are approximate as the decompositions did not reach50% in 168 hours ** The half life is calculated as ln(2)/rate constant.

Example 44 Water in Polymer Conjugates

Selected polymers were analyzed by Karl Fisher (QTI Salem IndustrialPark—Bldg. #5 Whitehouse, N.J. 08888) to determine the water content ofthe polymer. The results are summarized below in Table 2.

TABLE 2 Sample Water Constant % O-7175 6.56 O-7320 9.65 O-7271 6.71O-7376 5.13

Example 45 Reaction of Carbamoylfumagillol with 2-Mercaptopyrimidine

A stock solution, 1 mg/mL, of 2-mercaptopyrimidine (2.2 mL) inmethanol-D4 was added to carbamoylfumagillol (6.4 mg). One mL of theresulting solution was removed and a second portion of the stocksolution was added (1 mL). Solid K₂CO₃ was added and the solutionmonitored by ¹H NMR. A single product was identified, the 1:1 adduct of2-mercaptopyrimidine and carbamoylfumagillol.

The following resonances were used to monitor the reaction by ¹H NMR:

2-Mercaptopyrimidine showed resonances at 6.7 ppm (IH, H-4) and 8.1 ppm(2H, H-3, H-5).

The adduct of 2-mercaptopyrimidine showed resonances at 7.2 ppm (1H,H-4) and 8.5-8.6 ppm (2H, H-3, H-5).

Example 46 Reaction of Polymer Conjugates with 2-Mercaptopyrimidine

A stock solution, 1 mg/mL, of 2-mercaptopyrimidine (1.1 mL) inmethanol-D4 was added to the polymer conjugate (10 mg). The solution wasstirred at room temperature overnight, and analyzed by ¹H NMR todetermine the ratio of unreacted thiol (8.1 ppm) to reacted thiol(8.5-8.6 ppm). The amount of reacted thiol was expected to be equivalentto the quantity of fumagillol in the polymer conjugate. The acetamidecapped polymer containing no epoxide showed no reaction product with2-mercaptopyrimidine as indicated in Table 3.

TABLE 3 Sample Reacted thiol/g polymer O-7175 0.37 mmols/g O-7320 0.37mmols/g O-7376 <0.001 mmols/g   

Example 47 Cathepsin B Release of Fumagillol Analogs

Cathepsin B (Sigma Cat #C6286 Lot #025K7672) was diluted to a 10×concentration in activation buffer consisting of approximately 400 nMenzyme, 30 mM DTT, 15 mM EDTA and acetate buffer, pH=5.5 for 15 minutesat room temperature.

The HPMA conjugates were made into a 10× stock solution in pH 5.5buffer. The final reaction was performed by diluting the enzyme andsubstrate 10 fold into either buffer at pH=5.5 or pH=6.8. The finalenzymatic reaction consisted of 40 nM Cathepsin B, approximately 2.5mg/mL test agent, and buffer at 37° C. The reaction was stopped at 0, 2,6, and 24 hour. To stop the reaction, 3 volumes of ice-cold methanolcontaining propranolol internal standard (at 1.0 μM) was added and lefton ice. The samples were then analyzed by LC/MS/MS.

Poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol] was shown torelease N-(6-aminohexyl)carbamoylfumagillol and fumagil-6-yl{6-[(aminoacetyl)amino]hexyl}carbamate.

Poly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl) wasshown to release fumagillol, carbamoylfumagillol, and fumagil-6-yl(2-aminoethyl)methylcarbamate.

Poly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl) wasshown to release fumagillol, carbamoylfumagillol, fumagil-6-ylmethyl[2-(methylamino)ethyl]carbamate, and ethyl{2-[(aminoacetyl)(methyl)amino]ethyl}methylcarbamate.

Example 48 General Materials and Methods for In Vitro Analysis

Test compounds, small molecules or polymer conjugates, were dissolved indimethyl sulfoxide to a stock concentration of 5 mg/mL. The test agentswere then diluted to an intermediate concentration at 200 μg/mL in 10%DMSO. Further dilutions were completed serially 3-fold in 10% DMSO toproduce 12 decreasing concentrations for in-vitro analysis. To achievethe target concentrations of the in-vitro assays, 1 of the intermediatedrug preparation was delivered to the cells (seeded in a volume of 50μL). The final DMSO concentration for the tests was 0.2% for all dosesof test agent.

Cells were exposed to twelve increasing concentrations of formulatedtest agent from 2×10⁻⁶ to 4.0 μg/mL for 72 hours. Following 72 hourexposure, 25 μL of CellTiter-Glo® Reagent was added to each well. Theplates were incubated for 60 minutes at 37° C. in a humidifiedincubator. After incubation, luminescence was recorded using theMolecular Devices AnalystGT multi-mode reader.

IC50 Determination

Data are expressed as the percent cell growth of the untreated (vehicle)control calculated from the luminescence signals. The surviving fractionof cells is determined by dividing the mean luminescence values of thetest agents by the mean luminescence values of untreated control. Theinhibitory concentration value for the test agent(s) and control wereestimated using Prism 5 software (GraphPad Software, Inc.) bycurve-fitting the data using the non-linear regression analysis.

Example 49 A549 Human Non-Small Cell Lung Carcinoma Cell Viability Assay

The human tumor cell lines A549 and HCT-116 were obtained from AmericanType Culture Collection (Manassas, Va.). The Human umbilical veinepithelial cells (HUVEC) were obtained from Lonza (Basel, Switzerland).The A549 cells were maintained RPMI 1640 w/L-glut supplemented with 5%FBS. The HCT-116 cells were maintained in McCoy's 5a supplemented with5% FBS. The HUVEC line was grown in Endothelial Growth Medium withsupplements and growth factors (BBE, hydrocortisone, hEGF, FBS andgentamicin/amphotericin-B). All cells were house in an atmosphere of 5%CO₂ at 37° C. Cells were dissociated with 0.05% Trypsin and 0.02% EDTA.

The human tumor cell line A549 was obtained from American Type CultureCollection (Manassas, Va.). The A549 cells were maintained RPMI 1640w/L-glut supplemented with 5% FBS. A549 cells were seeded at 500 cellsper well 24 hours prior to test agent exposure in a volume of 50 μL. Thecells were housed in an atmosphere of 5% CO₂ at 37° C. Cells weredissociated with 0.05% Trypsin and 0.02% EDTA.

TABLE 4 Table 4. A549 - Small Molecules A549 IC50 Compound Average ng/mLCompound # 0.508 O-7233 0.777 O-7299 1.50 O-7322 5.99 O-7319 23.2 O-72870.215 O-7177 1.06 O-7216 Carbamoylfumagillol 2.89 O-7127-1 TNP-470 8.97O-7178 Fumagillol 30.1 O-7126-1

TABLE 5 Table 5. 549 - Polymer Conjugates A549 IC50 Compound Averageng/mL Compound # 0.86 O-7172 1.08 O-7173 0.40 O-7174 0.50 O-7175 2.57O-7176 1.11 O-7192 0.28 O-7193 1.12 O-7195 0.67 O-7196 0.12 O-7215 0.52O-7232 0.40 O-7234 1.16 O-7271 0.08 O-7272 0.17 O-7303 0.42 O-7304 4.00O-7305 0.89 O-7306 0.32 O-7320 0.42 O-7321 0.98 O-7323 1.54 DRS-226-46E

Example 50 HCT-116 Human Colon Tumor Cell Viability Assay

The human tumor cell lines A549 and HCT-116 were obtained from AmericanType Culture Collection (Manassas, Va.). The HCT-116 cells weremaintained in McCoy's 5a supplemented with 5% FBS. HCT-116 cells wereseeded at 500 cells per well 24 hours prior to test agent exposure in avolume of 50 μL. The cells were housed in an atmosphere of 5% CO₂ at 37°C. Cells were dissociated with 0.05% Trypsin and 0.02% EDTA.

Cells were exposed to twelve increasing concentrations of formulatedtest agent from 2.3×10⁻⁶ to 4.02 μg/mL for 72 hours. Following 72 hourexposure, 25 μL of CellTiter-Glo® Reagent was added to each well. Theplates were incubated for 60 minutes at 37° C. in a humidifiedincubator. After incubation, luminescence was recorded using theMolecular Devices AnalystGT multi-mode reader.

TABLE 6 Table 6. HCT116 - Small Molecules HCT116 IC50 Compound Averageng/mL Compound # 0.236 O-7177 0.408 O-7194 0.918 O-7216Carbamoylfumagillol 1.035 O-7127-1 TNP-470 2.64 O-7178 Fumagillol 45.8O-7216-1

TABLE 7 Table 7. HCT116 - Polymer Conjugates HCT116 IC50 CompoundAverage ng/mL Compound # 0.157 O-7215 0.329 O-7193 0.392 O-7174 0.626O-7175 0.818 O-7196 1.221 O-7172 1.051 O-7173 1.184 O-7192 1.203 O-71950.984 DRS-226-46E 5.954 O-7176

Example 51 Human Umbilical Vein Epithelial Cell Viability Assay

The Human umbilical vein epithelial cells (HUVEC) were obtained fromLonza (Basel, Switzerland). The HUVEC line was grown in EndothelialGrowth Medium with supplements and growth factors (BBE, hydrocortisone,hEGF, FBS and gentamicin/amphotericin-B). All cells were housed in anatmosphere of 5% CO₂ at 37° C. Cells were dissociated with 0.05% Trypsinand 0.02% EDTA.

HUVEC cells were seeded at 1000 cells per well 24 hours prior to testagent exposure in a volume of 50 μE. Cells were exposed to twelveincreasing concentrations of formulated test agent from 2.3×10⁻⁶ to 4.02μg/mL for 72 hours. Following 72 hour exposure, 25 of CellTiter-Glo®Reagent was added to each well. The plates were incubated for 60 minutesat 37° C. in a humidified incubator. After incubation, luminescence wasrecorded using the Molecular Devices AnalystGT multi-mode reader.

TABLE 8 Table 8. HUVEC - Small Molecules HUVEC IC50 Compound Averageng/mL Compound # 0.101 O-7177 0.120 O-7194 0.209 O-7216Carbamoylfumagillol 0.086 O-7127-1 TNP-470 0.153 O-7178 Fumagillol 18.9O-7126-1

TABLE 9 Table 9. HUVEC - Polymer Conjugates HUVEC IC50 Compound Averageng/mL Compound # 0.157 O-7215 0.329 O-7193 0.392 O-7174 0.626 O-71750.818 O-7196 1.221 O-7172 1.051 O-7173 1.184 O-7192 1.203 O-7195 0.984DRS-226-46E 5.954 O-7176

Example 52 A549/HUVEC Selectivity

The ratio of the HUVEC IC50/A549 IC₅₀ is presented in Table 10 below.When compared to carbamoylfumagillol and TNP-470, the polymer conjugatesare more active against the tumor cells, A549, than against the normalHUVEC cells.

TABLE 10 Compound A549/HUVEC IC50 Compound # IC50 ratio 2.14 O-7177 2.97O-7194 5.06 O-7216 Carbamoylfumagillol 33.63 O-7127-1 TNP-470 58.53O-7178 Fumagillol 1.59 O-7126-1 Polymer Conjugates 0.66 0-7215 3.130-7193 2.04 0-7174 2.64 0-7175 2.26 0-7196 1.52 0-7172 1.30 0-7173 1.810-7192 1.66 0-7195 4.33 DRS-226-46E 0.98 0-7176

Example 53 A549 Metabolites

Cells were treated as in Example 51 except that at the end of 72 hourexposure to test agent, the cells were frozen (−70° C.) and stored forsubsequent evaluation by LC/MS. Metabolites identified from the cellstreated with poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol]include N-(6-aminohexyl)carbamoylfumagillol,fumagill-6-yl{6-[(aminoacetyl)amino]hexyl}carbamate, and the epoxide hydrolysisproduct,(35′,7aR)-7a-(hydroxymethyl)-4-methoxy-3-methyl-2-(3-methylbut-2-en-1-yl)octahydro-1-benzofuran-3-ol-5-yl6-aminohexyl carbamate.

Example 54 In Vivo Testing B16-F 10 Murine Melanoma

C57B16 female mice (N=8) were injected (tail vein) with 1×10⁵ B16-F10tumor cells. After one day, mice were treated with polymer conjugates assolutions in saline (IV administration, q4d, four doses except that inone group 0-7175 was administered as a single dose on day 1). TNP-470was used as a positive control, saline as a negative control. Mice weresacrificed after 15 days. Treatment outcomes were assessed by countinglung metastases.

TABLE 11 Table 11. Metastases Counts Metastases Group Dose mg/kg* CountsSaline control 0 36.8 TNP-470 30 39.5 O-7175 50 17.0 O-7175 100 24.5O-7175 200 20.9 O-7320 200 7.6 O-7271 200 20.0 O-7215 200 32.5 O-71751000 10.1 *All groups, N = 8. IV dosing q4d, days 1, 5, 9 and 13 exceptTNP-470 (qod) and O-7175 at 1000 mg/kg (single dose on day 1).

Example 55 In Vivo Testing C57B16 Mice—Weight Changes

C57B16 female mice (N=8) were injected (tail vein) with 1×10⁵ B16-F10tumor cells. After one day, mice were treated with polymer conjugates assolutions in saline (IV administration, q4d, four doses). The weightchanges for three polymers relative to saline vehicle control andTNP-470 are shown in FIG. 1. Weight changes are referenced to the groupweight at time zero. All polymers were dosed at 100 mg/kg. Polymer dosesand the saline vehicle were administered on days 1, 5, and 9. The 100mg/kg polymer doses and TNP-470 showed a reduction in metastases from44-63% relative to the saline control.

Example 56 In Vivo Testing C57B16 Mice Weight Changes

C57B16 female mice (N=8) were injected (tail vein) with 1×10⁵ B16-F10tumor cells. After one day, mice were treated with polymer conjugates assolutions in saline (IV administration, q4d, four doses). The weightchanges for one polymer at three different doses relative to control areshown in FIG. 2. Weight changes are referenced to the group weight attime zero. The polymer doses were 50 mg/kg, or 100 mg/kg. Polymer doseswere administered on days 1, 5, and 9. The 25, 50 and 100 mg/kg polymerdoses and TNP-470 showed a reduction in metastases from 45-61% relativeto the saline control.

Example 57 In Vivo Testing nu/nu Mice—A 549 Xenograft

Nu/nu female mice (N=8) were injected (subcutaneous right flank) with5×10⁶ A549 tumor cells (inoculation vehicle 50% media/matrigel,subcutaneous right flank). After the tumors reached a size of 116 mg,mice were treated with polymer conjugates as solutions in saline (20mg/kg, IV administration, q4d, six doses) or with a control polymerwithout a MetAP2 inhibitory moiety (100 mg/kg, q4d) or with TNP-470 (30mg/kg, qod, nine doses). Tumor growth was determined by measuring tumorsize in two directions with calipers at intervals of a few days. Thetumor size vs time is shown in FIG. 3. The doses used are summarized inthe table below.

TABLE 12 Single Dose Total Dose Total Dose Schedule # doses mg/kg mgmmol active TNP-470 qod 9 30 270 0.67  Polymer Q4d 6 20 120 0.044frequency # doses wt/wt wt/wt mol/mol Polymer % 50% 67% 67% 44% 7%

The change in body weight vs time for the A549 Xenograft experiment isshown in FIG. 4. The mice in the active polymer treated groups showsimilar weight changes to the TNP-470 group and the control groups.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A pharmaceutical composition comprising thecompound:

wherein x is between 1 and about 450, y is between 1 and about 30, and nis between 1 and about 50, or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein the ratio of x:y is inthe range of about 15:1 to about 6:1.
 3. A pharmaceutical compositioncomprising the compound:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 4. A pharmaceutical composition comprising thecompound:

wherein x is between 1 and about 450, y is between 1 and about 30, and nis between 1 and about 50, or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier.
 5. Thepharmaceutical composition of claim 4, wherein the ratio of x:y is inthe range of about 15:1 to about 6:1.
 6. A pharmaceutical compositioncomprising the compound:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 7. A pharmaceutical composition comprising thecompound:

wherein x is between 1 and about 450, y is between 1 and about 30, and nis between 1 and about 50, or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition of claim 7, wherein the ratio of x:y is inthe range of about 15:1 to about 6:1.
 9. A pharmaceutical compositioncomprising the compound:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 10. A pharmaceutical composition comprising thecompound:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.